Method and system for reaction vessel with multisided energy sources

ABSTRACT

Embodiments include a reaction vessel having a reaction chamber defined by opposing first and second interior-facing surfaces of the housing; a first light absorbing layer conforming to the first interior-facing surface of the housing component; and a second light absorbing layer conforming to the second interior-facing surface of the housing component; a first energy source configured to direct light through the housing at the first light absorbing layer; and a second energy source configured to direct light through the housing at the second light absorbing layer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/746,488, filed Oct. 16, 2018, entitled “METHOD AND SYSTEM FORTEMPERATURE MONITORING OF A BIOCHEMICAL VESSEL,” and U.S. ProvisionalPatent Application No. 62/746,492, filed Oct. 16, 2018, entitled “METHODAND SYSTEM FOR REACTION VESSEL WITH MULTISIDED ENERGY SOURCES,” thedisclosures of which are hereby incorporated by reference in theirentirety for all purposes.

The following regular U.S. patent applications (including this one) arebeing filed concurrently, and the entire disclosure of the otherapplications are incorporated by reference into this application for allpurposes:

-   -   application Ser. No. ______, filed Oct. 16, 2018, entitled        “METHOD AND SYSTEM FOR REACTION VESSEL WITH MULTISIDED ENERGY        SOURCES” (Attorney Docket No. 104587-000310US-1156117); and    -   application Ser. No. ______, filed Oct. 16, 2018, entitled        “METHOD AND SYSTEM FOR TEMPERATURE MONITORING OF A BIOCHEMICAL        REACTION VESSEL” (Attorney Docket No. 104587-000410US-1156135).

BACKGROUND OF THE INVENTION

Reaction vessels are often used to perform various operations on DNAstrands that can include operations such as polymerase chain reaction(PCR) and DNA sequencing. Polymerase chain reaction (PCR) has become anessential technique in the fields of clinical laboratories, agriculturalscience, environmental science, and forensic science. PCR requiresthermal cycling, or repeated temperature changes between two or threediscrete temperatures to amplify specific nucleic acid target sequences.To achieve such thermal cycling, conventional bench-top thermal cyclersgenerally use a metal heating block powered by Peltier elements.Unfortunately, this method of thermally cycling the materials within thereaction vessels can be slower than desired. For these reasons,alternate means that improve the speed and/or reliability of the thermalcycling are desirable.

SUMMARY OF THE INVENTION

This disclosure relates to methods and apparatus suitable for use inreactions, assays, or experiments where temperature control ormonitoring is desired. A number of reaction vessels and systems aredisclosed, along with methods of operating the reaction vessels andsystems, and methods of manufacturing the reaction vessels.

In some embodiments, a reaction vessel system may include a reactionvessel comprising: a housing; a first reaction chamber defined byopposing first and second interior-facing surfaces of the housing; afirst light absorbing layer conforming to the first interior-facingsurface of the housing; and a second light absorbing layer conforming tothe second interior-facing surface of the housing. The reaction vesselsystem may further include a first energy source configured to directlight through the housing at the first light absorbing layer; and asecond energy source configured to direct light through the housing atthe second light absorbing layer. In some embodiments, the housing isconfigured to be disposed between the first energy source and the secondenergy source when in use. In some embodiments, the first lightabsorbing layer and the second light absorbing layer have a samethickness and composition.

In some embodiments, the first and second light absorbing layers eachcomprise a metallic film formed on respective first and secondinterior-facing surfaces of the housing. In some embodiments, themetallic film is formed on, deposited on, adhered to, or otherwisedisposed on the first and second interior-facing surfaces of thehousing.

In some embodiments, at least a portion of the housing is opticallytransparent to wavelengths of light emitted by the first and secondenergy sources. In some embodiments, the first energy source is a lightemitting diode configured to emit visible light. In some embodiments,the first energy source is a light emitting diode configured to emitinfrared light.

In some embodiments, the reaction vessel system may include anexcitation light source assembly and an emission detecting sensorassembly, wherein the excitation light source assembly comprises anexcitation light source configured to direct an excitation lightconfigured to cause a fluorescent marker within the first reactionchamber to emit a fluorescent light, and wherein the emission detectingsensor assembly comprises an emission sensor configured to detect theemitted fluorescent light. The reaction vessel system may include anemission filter disposed between the first reaction chamber and theemission sensor, wherein the emission filter is configured to allowlight of one or more first wavelengths corresponding to the emittedfluorescent light, and filter out light of one or more secondwavelengths, wherein the one or more first wavelengths are differentfrom the one or more second wavelengths. The reaction vessel system mayinclude an excitation filter disposed between the first reaction chamberand the excitation light source, wherein the excitation filter isconfigured to allow light of one or more third wavelengths configured toexcite the fluorescent marker, and filter out light of one or morefourth wavelengths, wherein the one or more third wavelengths aredifferent from the one or more fourth wavelengths.

In some embodiments, the first and second energy sources furthercomprise optical fibers that carry the emitted fluorescent light atleast a portion of a distance between the energy sources and thehousing. In some embodiments, the first energy source comprises a chipon board LED (COB LED), wherein the COB LED comprises a plurality of LEDchips that are configured to be individually controllable.

In some embodiments, the reaction vessel system includes a secondreaction chamber; a third energy source configured to direct lightthrough the housing at the first light absorbing layer adjacent to thesecond reaction chamber; and a fourth energy source configured to directlight through the housing at the second light absorbing layer adjacentto the second reaction chamber; wherein the first, second, third, andfourth energy sources are individually controllable. In someembodiments, the first light absorbing layer comprises a first discreteregion associated with the first reaction chamber and a second discreteregion associated with the second reaction chamber, and wherein thefirst energy source is configured to direct light at the first discreteregion and the third energy source is configured to direct light at thesecond discrete region.

In some embodiments, the first energy source and the second energysource are optical fibers coupled to a single precursor energy source,wherein energy from the single precursor energy source is dividedbetween the first energy source and the second energy source.

In some embodiments, the first and second light absorbing layers havingan inner surface that is oriented toward an interior of the firstreaction chamber and an outer surface that is oriented away from theinterior of the first reaction chamber, and wherein the first and secondenergy sources are configured to direct light at the outer surfaces ofthe first and second light absorbing layers

A method of operating a temperature-controlled reaction vessel system isdisclosed, wherein the method includes: introducing a reagent into afirst reaction chamber, wherein the first reaction chamber comprises afirst light absorbing layer and a second light absorbing layer, thefirst and second light absorbing layers having an inner surface that isoriented toward an interior of the first reaction chamber and an outersurface that is oriented away from the interior of the first reactionchamber; direct, by a first energy source, a first light toward theouter surface of the first light absorbing layer so as to heat the firstlight absorbing layer; direct, by a second energy source, a second lighttoward the outer surface of the second light absorbing layer so as toheat the second light absorbing layer; and causing heat from the firstand second light absorbing layers to be transferred to the reagent.

In some embodiments, the method includes introducing a reagent into asecond reaction chamber; causing a third energy source to direct a thirdlight toward an outer surface of the first light absorbing layeradjacent to a second reaction chamber; and causing a fourth energysource to direct a fourth light toward an outer surface of the secondlight absorbing layer adjacent to the second reaction chamber.

In some embodiments, the first light absorbing layer comprises a firstdiscrete region associated with the first reaction chamber and a seconddiscrete region associated with the second reaction chamber, and whereinthe first light is directed toward the first discrete region and thethird light is directed toward the second discrete region. In someembodiments, the second light absorbing layer comprises a first discreteregion associated with the first reaction chamber and a second discreteregion associated with the second reaction chamber, and wherein thesecond light is directed toward the first discrete region and the fourthlight is directed toward the second discrete region.

In some embodiments, the method includes directing an excitation lightfrom an excitation light source toward the first reaction chamber,wherein the excitation light is configured to cause a fluorescent markerwithin the first reaction chamber to emit a fluorescent light; anddetecting, by an emission detecting sensor assembly, the emittedfluorescent light. The method may include filtering light reaching theemission detecting sensor assembly with an emission filter disposedbetween the first reaction chamber and the emission sensor, wherein thefiltering comprises allowing light of one or more first wavelengthscorresponding to the emitted fluorescent light, and filtering out lightof one or more second wavelengths, wherein the one or more firstwavelengths are different from the one or more second wavelengths. Themethod may include filtering light from the excitation light source withan excitation filter disposed between the first reaction chamber and theexcitation light source, wherein the filtering comprises allowing lightof one or more third wavelengths configured to excite the fluorescentmarker, and filtering out light of one or more fourth wavelengths,wherein the one or more third wavelengths are different from the one ormore fourth wavelengths.

In some embodiments, the method may include detecting, by a temperaturesensor, a temperature associated with the first reaction chamber; andlogging, within a memory, a value indicating the detected temperatureand a value corresponding to the emitted fluorescent light (e.g., avalue quantifying the amount of emitted fluorescent light, or simplyindicating whether or not a threshold amount of fluorescent light wasemitted).

In some embodiments, a reaction vessel system may include a firstchamber filled with a first material; a first light absorbing regionadhered to a first interior-facing surface of the first chamber; asecond chamber filled with a second material, wherein the secondmaterial is different from the first material; a second light absorbingregion adhered to a first interior-facing surface of the second chamber;a temperature sensor disposed within the second chamber for measuring asecond temperature; one or more energy sources configured to directlight at the first light absorbing region and the second light absorbingregion; and a processor configured to determine a first temperature ofthe first chamber based on the second temperature of the second chambermeasured by the temperature sensor.

In some embodiments, the reaction vessel system includes one or moreenergy attenuating features configured to reduce an amount of energytransmitted to the second light absorbing region. In some embodiments,the one or more energy attenuating features are selected from a groupconsisting of a light diffusing layer, a light reflecting layer, afilter layer, or a light blocking layer.

In some embodiments, the one or more energy sources comprise a firstenergy source configured to direct light at the first light absorbingregion and a second energy source configured to direct light at thesecond light absorbing region.

In some embodiments, the first material has a different specific heatthan the second material. In some embodiments, the second material is apolymeric material, an adhesive material, or any other suitablematerial.

In some embodiments, the reaction vessel system includes an excitationlight source assembly and an emission detecting sensor assembly, whereinthe excitation light source assembly comprises an excitation lightsource configured to direct an excitation light configured to cause afluorescent marker within the first chamber to emit a fluorescent light,and wherein the emission detecting sensor assembly comprises an emissionsensor configured to detect the emitted fluorescent light.

In some embodiments, the reaction vessel system includes a third lightabsorbing region adhered to a second interior-facing surface of thefirst chamber, wherein the second interior-facing surface of the firstchamber opposes the first interior-facing surface of the first chamber;a fourth light absorbing region adhered to a second interior-facingsurface of the second chamber, wherein the second interior-facingsurface of the first chamber opposes the first interior-facing surfaceof the first chamber; and one or more additional energy sourcesconfigured to direct light at the third light absorbing region and thefourth light absorbing region.

In some embodiments, a reaction vessel may include a first chamberfilled with a first material; a first light absorbing region adhered toa first interior-facing surface of the first reaction chamber; a secondchamber filled with a second material, wherein the second material isdifferent from the first material; a second light absorbing regionadhered to a first interior-facing surface of the second chamber; atemperature sensor disposed within the second chamber for measuring asecond temperature; and a connector for coupling the temperature sensorto a processor, wherein the temperature sensor is configured to send asignal corresponding to the second temperature to the processor fordetermining a correlated first temperature corresponding to the firstchamber. The first light absorbing region and the second light absorbingregion may be configured to absorb energy from one or more lightsources. In some embodiments, reaction vessel may include one or moreenergy attenuating features configured to reduce an amount of energytransmitted to the second light absorbing region.

In some embodiments, a method of monitoring a reaction chambertemperature is disclosed. The method may include measuring a firsttemperature of a temperature-monitoring chamber associated with areaction vessel, wherein the temperature-monitoring chamber is filledwith a potting material; transmitting a signal corresponding to thefirst temperature to a processor; determining, by the processor, a firsttemperature value corresponding to the first temperature; andestimating, based on the first temperature value, a second temperaturevalue associated with a reaction chamber. In some embodiments, thetemperature-monitoring chamber is housed in a module that is separatedfrom the reaction vessel.

In some embodiments, the estimating comprises accessing a lookup tablecorrelating a plurality of temperature values corresponding to thetemperature-monitoring chamber with a plurality of temperature valuescorresponding to the reaction chamber. In some embodiments, theestimating comprises applying a function to the first temperature valueto generate the second temperature value.

In some embodiments, the potting material has a different specific heatthan a material within the reaction chamber.

In some embodiments, the method includes adjusting, based on the firsttemperature, a power level of an energy source configured to heat thereaction chamber.

In some embodiments, a method of manufacturing a reaction vessel isdisclosed. The method may include forming a first portion of a reactionvessel housing, the first portion of the reaction vessel housing havinga plurality of depressions; forming a second portion of the reactionvessel housing; securing the first and second portions of the reactionvessel housing to each other, such that the depressions at least in partdefine a plurality of chambers, the plurality of chambers comprising areaction chamber and a temperature-monitoring chamber; introducing aliquid material into the temperature-monitoring chamber; placing atemperature sensor at a desired position within thetemperature-monitoring chamber; and causing the liquid material tosolidify within the temperature-monitoring chamber.

In some embodiments, the liquid material is a polymeric material. Insome embodiments, the liquid material is a silane-modified polymer. Inthese examples, the liquid material may be caused to solidify bydirecting UV energy at the temperature-monitoring chamber.

In some embodiments, the second portion of the reaction vessel housinghas one or more depressions.

In some embodiments, reaction vessel may include a housing having aplurality of walls that define a reaction chamber filled with a solution(e.g., one or more chemicals or biologicals, reagents); a lightabsorbing region adhered to an interior-facing surface of a first wallof the plurality of walls defining the reaction chamber; a temperaturesensor embedded within a second one of the plurality of walls definingthe reaction chamber; an energy source configured to direct lightthrough the reaction chambers at the light absorbing region. In someembodiments, a temperature of the solution may be determined (e.g., by aprocessor) based on a measurement made by the temperature sensor. Insome embodiments, the first wall is different from the second wall.

Other aspects and advantages of the invention will become apparent fromthe following detailed description taken in conjunction with theaccompanying drawings which illustrate, by way of example, theprinciples of the described embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure will be readily understood by the following detaileddescription in conjunction with the accompanying drawings, wherein likereference numerals designate like structural elements, and in which:

FIG. 1A shows an exemplary reaction vessel suitable for use with thedescribed embodiments;

FIG. 1B shows another exemplary reaction vessel suitable for use withthe described embodiments;

FIG. 1C shows how air gap regions establish robust barriers that reducethe lateral transfer of heat between adjacent reaction vessels;

FIG. 1D shows housing components arranged in a hexagonal pattern withone of the housing components having a temperature sensor disposedwithin its reaction chamber;

FIG. 2A shows a cross-sectional schematic view of an exemplary reactionvessel including a light absorbing layer that can take the form of athin metallic film;

FIG. 2B shows how two light absorbing layers can be positioned upon oradjacent to opposing interior-facing surfaces of housing, which in partdefine reaction chamber;

FIG. 3A shows an example configuration of light absorbing layers and canbe positioned upon or adjacent to opposing interior-facing surfaces ofhousing with optical elements and that may be used to alter thecharacteristics of light generated by energy sources;

FIG. 3B shows how multiple reaction chambers can be positioned within ahousing of a reaction vessel;

FIG. 4 shows an example embodiment in which an energy source takes theform of a chip on board (COB) LED that may be well suited fordistributing light evenly across a relatively large area of housing ofreaction vessel;

FIG. 5A shows an example embodiment with a PCB supporting multiple LEDsthat can be used to illuminate opposing sides of a reaction chamber ofreaction vessel;

FIG. 5B shows an example embodiment where light energy from a singleprecursor energy source (e.g., the LED) is split between two opticalfibers to illuminate opposing sides of a reaction chamber of reactionvessel;

FIG. 5C shows the energy source configuration depicted in FIG. 5A withthe addition of an excitation light source assembly and an emissiondetecting sensor assembly;

FIGS. 6A-6B show experimental data from three consecutive heating andcooling test cycles comparing temperature profiles of a reaction chamberbeing heated from one side (one-sided heating) and a reaction chamberbeing heated from two sides (dual-sided heating);

FIG. 7 illustrates an example method for operating atemperature-controlled reaction vessel system;

FIG. 8A shows an example reaction vessel with a housing that definesmultiple reaction chambers that can be filled with solution and achamber that includes a temperature sensor;

FIG. 8B shows a reaction vessel that includes a housing defining atleast two reaction chambers, a chamber that includes a temperaturesensor, and a light diffusing layer;

FIG. 8C shows a reaction vessel having a housing defining a chamber thatis different from the reaction chambers;

FIG. 8D shows an example reaction vessel having a light diffusion layerand a light reflecting layer;

FIG. 8E shows an embodiment in which a temperature sensor can beembedded within a housing of a reaction vessel;

FIG. 8F shows an example embodiment in which temperature sensor isdisposed within a module that is separate and distinct from a housing ofa reaction vessel;

FIG. 9A shows a lateral schematic view of a cross-section of an examplereaction vessel with a chamber containing a temperature sensor;

FIG. 9B shows an overhead schematic view of a cross-section of theexample reaction vessel depicted in FIG. 9A with a chamber having atemperature sensor coupled to a processor;

FIG. 10 shows a simplified schematic diagram of a reaction vesselsystem;

FIG. 11A shows experimental data from tests conducted on apolymer-filled chamber and a water-filled chamber;

FIG. 11B shows experimental data from 24 successive PCR for 45 cyclesusing a polymer-filled reference chamber as a temperature-monitoringchamber with reference to which the different steps of PCR cycles wereperformed for all 24 successive PCR;

FIGS. 11C-11D show experimental data from PCR performed using apolymer-filled temperature monitoring chamber as a reference, withdifferent initial DNA template concentrations;

FIG. 12 shows a block diagram illustrating a method for assembling atemperature monitoring assembly;

FIG. 13 illustrates an example method for manufacturing a reactionvessel; and

FIG. 14 illustrates an example method for monitoring a reaction chambertemperature.

DETAILED DESCRIPTION OF THE INVENTION

In the following detailed description, references are made to theaccompanying drawings, which form a part of the description and in whichare shown, by way of illustration, specific embodiments in accordancewith the described embodiments. Although these embodiments are describedin sufficient detail to enable one skilled in the art to practice thedescribed embodiments, it is understood that these examples are notlimiting; such that other embodiments may be used, and changes may bemade without departing from the spirit and scope of the describedembodiments.

Microfluidics systems or devices have widespread use in chemistry andbiology. In such devices, fluids are transported, mixed, separated orotherwise processed. In many microfluidics devices, various applicationsrely on passive fluid control using capillary forces. In otherapplications, external actuation means (e.g., rotary drives) are usedfor the directed transport of fluids. “Active microfluidics” refers tothe defined manipulation of the working fluid by active (micro)components such as micropumps or microvalves. Micropumps supply fluidsin a continuous manner or are used for dosing. Microvalves determine theflow direction or the mode of movement of pumped liquids. Processes thatare normally carried out in a laboratory can be miniaturized on a singlechip in order to enhance efficiency and mobility as well to reducesample and reagent volumes. Microfluidic structures can includemicropneumatic systems, i.e., microsystems for the handling of off-chipfluids (liquid pumps, gas valves, etc.), and microfluidic structures forthe on-chip handling of nanoliter (nl) and picoliter (pl) volumes(Nguyen and Wereley, Fundamentals and Applications of Microfluidics,Artech House, 2006).

Advances in microfluidics technology are revolutionizing molecularbiology procedures for enzymatic analysis (e.g., glucose and lactateassays), DNA analysis (e.g., polymerase chain reaction andhigh-throughput sequencing), and proteomics. Microfluidic biochipsintegrate assay operations such as detection, as well as samplepre-treatment and sample preparation on one chip (Herold and Rasooly,editors, Lab-on-a-Chip Technology: Fabrication and Microfluidics,Caister Academic Press, 2009; Herold and Rasooly, editors, Lab-on-a-ChipTechnology: Biomolecular Separation and Analysis, Caister AcademicPress, 2009). An emerging application area for biochips is clinicalpathology, especially the immediate point-of-care diagnosis of diseases.In addition, some microfluidics-based devices are capable of continuoussampling and real-time testing of air/water samples for biochemicaltoxins and other dangerous pathogens.

Many types of microfluidic architectures are currently in use andinclude open microfluidics, continuous-flow microfluidics, droplet-basedmicrofluidics, digital microfluidics, paper-based microfluidics and DNAchips (microarrays).

In open microfluidics, at least one boundary of the system is removed,exposing the fluid to air or another interface (i.e., liquid) (Berthieret al., Open microfluidics, Hoboken, N.J.: Wiley, Scrivener Publishing,2016; Pfohl et al., Chem Phys Chem. 4:1291-1298, 2003; Kaigala et al.,Angewandte Chemie International Edition. 51:11224-11240, 2012).Advantages of open microfluidics include accessibility to the flowingliquid for intervention, larger liquid-gas surface area, and minimizedbubble formation (Berthier et al., Open microfluidics, Hoboken, N.J.:Wiley, Scrivener Publishing, 2016; Kaigala et al., Ange. Chemie Int. Ed.51:11224-11240, 2012; Li et al., Lab on a Chip 17: 1436-1441). Anotheradvantage of open microfluidics is the ability to integrate open systemswith surface-tension driven fluid flow, which eliminates the need forexternal pumping methods such as peristaltic or syringe pumps (Casavantet al., Proc. Nat. Acad. Sci. USA 110:10111-10116, 2013). Openmicrofluidic devices are also inexpensive to fabricate by milling,thermoforming, and hot embossing (Guckenberger et al., Lab on a Chip,15: 2364-2378, 2015; Truckenmuller et al., J. Micromechanics andMicroengineering, 12: 375-379, 2002; Jeon et al., Biomed. Microdevices13: 325-333, 2010; Young et al., Anal. Chem. 83:1408-1417, 2011). Inaddition, open microfluidics eliminates the need to glue or bond a coverfor devices which could be detrimental for capillary flows. Examples ofopen microfluidics include open-channel microfluidics, rail-basedmicrofluidics, paper-based, and thread-based microfluidics (Berthier etal., Open microfluidics, Hoboken, N.J.: Wiley, Scrivener Publishing,2016; Casavant et al., Proc. Nat. Acad. Sci. USA 110:10111-10116, 2013;Bouaidat et al., Lab on a Chip 5: 827, 2005).

Continuous flow microfluidics are based on the manipulation ofcontinuous liquid flow through microfabricated channels (Nguyen et al.,Micromachines 8:186, 2017; Antfolk and Laurell, Anal. Chim. Acta965:9-35, 2017). Actuation of liquid flow is implemented either byexternal pressure sources, external mechanical pumps, integratedmechanical micropumps, or by combinations of capillary forces andelectrokinetic mechanisms. Continuous-flow devices are useful for manywell-defined and simple biochemical applications and for certain taskssuch as chemical separations, but they are less suitable for tasksrequiring a high degree of flexibility or fluid manipulations. Processmonitoring capabilities in continuous-flow systems can be achieved withhighly sensitive microfluidic flow sensors based onmicro-electro-mechanical systems (MEMS) technology, which offersresolutions down to the nanoliter range.

Droplet-based microfluidics manipulates discrete volumes of fluids inimmiscible phases with low Reynolds number and laminar flow regimes (seereviews at Shembekar et al., Lab on a Chip 8:1314-1331, 2016; Zhao-Miaoet al., Chinese J. Anal. Chem. 45:282-296, 2017. Microdroplets allow forthe manipulation of miniature volumes (μl to fl) of fluids conveniently,provide good mixing, encapsulation, sorting, and sensing, and aresuitable for high throughput applications (Chokkalingam et al., Lab on aChip 13:4740-4744, 2013).

Alternatives to closed-channel continuous-flow systems include openstructures, wherein discrete, independently controllable droplets aremanipulated on a substrate using electrowetting. By using discreteunit-volume droplets (Chokkalingam et al., Appl. Physics Lett.93:254101, 2008), a microfluidic function can be reduced to a set ofrepeated basic operations, i.e., moving one unit of fluid over one unitof distance. This “digitization” method facilitates the use of ahierarchical, cell-based approach for microfluidic biochip design.Therefore, digital microfluidics offers a flexible, scalable systemarchitecture as well as high fault-tolerance. Moreover, because eachdroplet can be controlled independently, these systems also have dynamicreconfigurability, whereby groups of unit cells in a microfluidic arraycan be reconfigured to change their functionality during the concurrentexecution of a set of bioassays. Alternatively, droplets can bemanipulated in confined microfluidic channels. One common actuationmethod for digital microfluidics is electrowetting-on-dielectric (EWOD)(reviewed in Nelson and Kim, J. Adhesion Sci. Tech., 26:12-17,1747-1771, 2012). Many lab-on-a-chip applications have been demonstratedwithin the digital microfluidics paradigm using electrowetting. However,recently other techniques for droplet manipulation have also beendemonstrated using magnetic force (Zhang and Nguyen, Lab on a Chip 17.6:994-1008, 2017), surface acoustic waves, optoelectrowetting, mechanicalactuation (Shemesh et al., Biomed. Microdevices 12:907-914, 2010), etc.

Paper-based microfluidics (Berthier et al., Open Microfluidics, JohnWiley & Sons, Inc. pp. 229-256, 2016) rely on the phenomenon ofcapillary penetration in porous media. In order to tune fluidpenetration in porous substrates such as paper in two and threedimensions, the pore structure, wettability and geometry of themicrofluidic devices can be controlled, while the viscosity andevaporation rate of the liquid play a further significant role. Manysuch devices feature hydrophobic barriers on hydrophilic paper thatpassively transport aqueous solutions to outlets where biologicalreactions take place (Galindo-Rosales, Complex Fluid-Flows inMicrofluidics, Springer, 2017).

Early biochips were based on the idea of a DNA microarray, e.g., theGeneChip DNA array from Affymetrix, which is a piece of glass, plasticor silicon substrate on which DNA molecules (probes) are affixed in anarray. Similar to a DNA microarray, a protein array is an array in whicha multitude of different capture agents, e.g., monoclonal antibodies,are deposited on a chip surface. The capture agents are used todetermine the presence and/or amount of proteins in a biological sample,e.g., blood. For a review, see, e.g., Bumgarner, Curr. Protoc. Mol.Biol. 101:22.1.1-22.1.11, 2013.

In addition to microarrays, biochips have been designed fortwo-dimensional electrophoresis, transcriptome analysis, and PCRamplification. Other applications include various electrophoresis andliquid chromatography applications for proteins and DNA, cellseparation, in particular, blood cell separation, protein analysis, cellmanipulation and analysis including cell viability analysis andmicroorganism capturing.

Reaction vessels are often used to perform various types of operationson DNA strands that include polymerase chain reactions (PCR) and DNAsequencing. Reaction vessels can incorporate one or more of themicrofluidics architectures listed above but it should be appreciatedthat reaction vessels can be larger than microfluidics devices and forthat reason may not incorporate any of the microfluidics architecturesdescribed above. Operations of the reaction vessels often include theneed to make rapid changes in temperature within the reaction vessel.For example, in a PCR operation solution containing DNA strands ispositioned within a reaction chamber defined by the reaction vessel. Aheating element is used to thermally cycle the solution in order tobreakdown and/or build up various different types of DNA. Unfortunately,conventional means of thermally cycling the solution are often slowerthan desired and not capable of varying a temperature of specificregions of a reaction chamber within the reaction vessel.

One solution to this problem is to position a light absorbing layerwithin the reaction chamber of the reaction vessel with light absorptioncharacteristics (e.g., allowing absorption of between 50 and 90% of thephotonic energy in any light absorbed by the light absorbing layer). Anenergy source can be configured to direct light at the light absorbinglayer, which efficiently absorbs energy from photons of the lightdirected at the light absorbing layer. The absorption of the photonicenergy may rapidly increase the temperature of the light absorbinglayer. This energy received by the light absorbing layer may then betransferred to a solution within the reaction chamber by thermalconduction and/or convection.

In some embodiments, a light absorbing layer extends across both upperand lower surfaces of a reaction chamber. This configuration allows thelight absorbing layer to have a larger area across which light can bereceived by one or more energy sources. For example, the energy sourcescan be positioned both above and below the reaction chamber. While thelight absorbing layer could be extended across any of the walls formingthe reaction chamber, extending the light absorbing layer across boththe upper and lower surface will generally double an amount of materialintroducing heat into a solution (e.g., one or more reagents) within thereaction chamber. The described configuration also reduces a thermalgradient formed within the reaction chamber since the solution can beheated from both the upper and lower surfaces, making the center of thereaction chamber likely to be the slowest in increasing in temperature.

In some embodiments, an array of reaction vessels or reaction chamberscan be grouped together and a temperature-monitoring module can replaceone of the reaction vessels or reaction chambers in order to accuratelymonitor a temperature of the reaction vessels or reaction chambersmaking up the array. The temperature-monitoring module can have asimilar size and shape to the reaction vessels or reaction chambers itreplaces. The temperature-monitoring module can include a temperaturesensor such as, e.g., a thermocouple, thermistor or resistancetemperature detector (RTD). The temperature sensor can be suspended in amaterial having a specific heat similar to a specific heat of thesolution within the other reaction vessels or reaction chambers. In thisway, the material within the temperature-monitoring module can closelymatch the temperature within the other reaction vessels or reactionchambers.

One benefit of this configuration is that this alleviates the need tosuspend a temperature sensor within reaction vessels or reactionchambers being used to perform various chemical/biological operations.The material can also be a polymeric material that is able to maintainthe temperature sensor at a central position within the reaction vesselor reaction chamber. In some embodiments, this can be preferable toplacing the temperature sensor within an active reaction vessel orreaction chamber where the temperature might be secured directly to alight absorbing layer that could result in readings from the temperaturesensor not reflecting the average temperature within the reaction vesselor reaction chamber.

These and other embodiments are discussed below; however, those skilledin the art will readily appreciate that the detailed description givenherein with respect to these figures is for explanatory purposes onlyand should not be construed as limiting.

FIG. 1A shows a perspective view of an exemplary reaction vessel 100suitable for use with the described embodiments. In particular, reactionvessel 100 includes a housing component 102 formed from an opticallytransparent material that defines a reaction chamber 104. While reactionchamber 104 is depicted as having a substantially circular geometry itshould be appreciated that the depicted shape of reaction chamber 104should not be construed as limiting and other shapes such as oval,rhombic and rectangular are also possible. In some embodiments, theoptically transparent material forming housing component 102 can beoptically transparent to only those wavelengths of light that are usedto heat reaction vessel 100. For example, the optically transparentmaterial could be optically transparent to only select visible, infraredor ultraviolet frequencies of light. Reaction chamber 104 can be closedby a second housing component (not depicted) that encloses a liquidbeing heated within reaction chamber 104. In this way, DNA strands in aliquid solution within reaction chamber 104 can undergo rapid thermalcycles and at least a portion of any vaporized portion of the solutioncan subsequently condense back into the solution between the thermalcycles or after the thermal cycling is complete. A light absorbing layer106 can be formed on, deposited on, adhered to, or otherwise disposed onan interior-facing surface of reaction chamber 104. Light absorbinglayer 106 has good light absorbing properties and can be in directcontact with any liquid disposed within reaction chamber 104. Forexample, light absorbing layer 106 can be configured to absorb about50-90% of the photonic energy incident to light absorbing layer 106. Insome embodiments, light absorbing layer 106 can be a metal film formedfrom elemental gold, chromium, titanium, germanium or a gold alloy suchas, e.g., gold-germanium, gold-chromium, gold-titanium,gold-chromium-germanium and gold-titanium-germanium. In someembodiments, light absorbing layer 106 can be a multilayer metal filmformed from elemental gold, chromium, titanium, germanium or a goldalloy such as, e.g., gold-germanium, gold-chromium, gold-titanium,gold-chromium-germanium and gold-titanium-germanium. Light absorbinglayer 106 can have a thickness of about 5 nm-200 nm. Housing component102 also defines inlet channel 108 and outlet channel 110, which can beused to cycle various chemicals, primers, DNA strands and otherbiological materials into and out of reaction chamber 104. In someembodiments, housing component 152 can have dimensions of about 7 mm by14 mm; however, it should be appreciated that this size can vary.

FIG. 1B shows a perspective view of another exemplary reaction vessel150. Reaction vessel 150, similar to reaction vessel 100 includeshousing component 152, reaction chamber 104, light absorbing layer 106,inlet channel 108 and outlet channel 110. Device housing 152 includes awidened central region that accommodates the inclusion of air gapregions 154 and 156. Air gap regions 154 and 156 can be left empty inorder to discourage the lateral transmission of heat to adjacentreaction vessels. In some embodiments, the transfer of heat through airgap regions 154 and 156 can be further reduced by removing the air fromair gap regions 154 and 156. In some embodiments, a diameter of housingcomponent 152 can be about 5 mm; however, it should be appreciated thatthis size can vary. For example, the diameter of housing component 152could vary from between 2 mm to 15 mm.

FIG. 1C shows how the shape of housing component 152 allow reactionvessels 150 to be packed tightly into a honeycomb or hexagonal pattern.FIG. 1C also illustrates how air gap regions 154 and 156 are able toestablish robust barriers that reduce the lateral transfer of heatbetween adjacent reaction vessels 150. When a diameter of reactionvessel 150 is about 5 mm reaction chamber 104 can hold about 10 μl ofsolution and have a depth of 800 μm. Generally, these devices areconfigured to hold between 2.5 μl and 500 μl with a depth of 200-1500μm.

FIG. 1D shows housing components 152 arranged in a hexagonal patternwith one of housing components 152 having a temperature sensor 158disposed within chamber 805. Temperature sensor 158 may be incorporatedwithin the chamber 805, and may be used to measure the heat introducedinto chamber 805 by light absorbing layer 106 along with minimal amountsof laterally-flowing heat transfer from adjacent reaction chambers 104.A temperature measured by temperature sensor 158 within chamber 805 maybe used to approximate a temperature of solution in nearby reactionchambers 104, as described in further detail below (e.g., in associatedwith FIGS. 8A-8F).

FIG. 2A shows a cross-sectional schematic view of an example reactionvessel 200. As illustrated, the reaction vessel 200 may include areaction chamber 104 and a light absorbing layer 106 disposed within ahousing 202. The light absorbing layer 106 may be disposed in a locationthat is adjacent to the reaction chamber 104. For example, the lightabsorbing layer 106 can take the form of a thin metallic film adhered toan interior-facing surface of housing 202 (e.g., light absorbing layer106 may be formed on, deposited on, adhered to, or otherwise disposed onan interior-facing surface of the reaction chamber 104). As anotherexample, the light absorbing layer 106 may be disposed adjacent to thereaction chamber 104, but may be covered with a substrate. In theexample configuration shown in FIG. 2A, light absorbing layer 106 coversonly one surface (e.g., a bottom surface) of reaction chamber 104. Thisconfiguration may allow for a large portion of the light emitted byenergy source 204 to be absorbed by light absorbing layer 106. Theenergy from the photons making up the light are converted into heatenergy, which is then thermally conducted into a solution withinreaction chamber 104.

In some embodiments, heating the solution within reaction chamber 104having a light absorbing layer 106 adjacent to only one interior-facingsurface may generate a thermal gradient within reaction chamber 104 froma portion of the solution farthest from the light absorbing layer 106 toa portion of the solution that is proximate to the light absorbing layer106. For example, a first portion of the solution farthest from lightabsorbing layer 106 may be heated more slowly than a second portion ofthe solution that is proximate to the light absorbing layer 106. In someembodiments, an operator may want to perform thermal cycles particularlyquickly, in which case, DNA or other chemicals located within the firstportion of the solution (e.g., farthest from light absorbing layer 106)may not be heated sufficiently quickly, and may thereby result in wastedmaterial and/or lower yields. Alternatively or additionally, the secondportion of the solution (e.g., proximate to the light absorbing layer106) may be overheated, which may result in some amount of bleachingoccurring to the materials within the solution, which can alsonegatively affect results of the operation. In part for this reason, insome embodiments it can be beneficial to illuminate opposing sides ofthe reaction vessel with two or more different energy sources (e.g.,multiple LEDs, a single LED whose emitted energy is split into multipleparts to effectively create multiple sources from the point of view ofthe reaction vessel). This could be implemented by adding a second lightabsorbing layer to the opposite side of the reaction chamber. As will befurther explained below, adding a second light absorbing layer increasesuniformity of heating, increases the overall speed of heating, andincreases the energy efficiency of heating.

FIG. 2B shows how two light absorbing layers 106-1 and 106-2 can bepositioned upon or adjacent to opposing interior-facing surfaces ofhousing 202, which in part define reaction chamber 104. Light absorbinglayers 106 can be configured to receive light from energy sources 204-1and 204-2 (e.g., LEDs) positioned upon opposing sides of reaction vessel200. Since this configuration allows heat to be introduced across a muchlarger area and on opposing sides of the reaction chamber, a solutionwithin the reaction chamber 104 may experience a much smaller thermalgradient as compared to the example reaction chamber of FIG. 2A (whichis heated from only one side). As a result, heating of the solution inthe configuration illustrated in FIG. 2B may be more uniform than in theconfiguration illustrated in FIG. 2A. For this reason, over- orunder-heating of the solution within reaction chamber 104 at any givenpoint may be less likely. Additionally, heating the solution frommultiple sides (e.g., from two opposing sides as shown in FIG. 2B) mayresult in faster heating of the solution, and as a result, may increaseoverall throughput for the reaction vessel. For example, a maximum rateat which the solution within reaction chamber 104 is thermally cycledcan be increased when compared to the maximum rate achievable byilluminating only a single side of the configuration illustrated in FIG.2A. This increase of the thermal cycling rate is at least in part aresult of being able to double the surface area receiving photonicenergy and the volume of heated film within reaction chamber 104. Insome embodiments, at least a portion of other interior-facing surfacescan be covered in a light absorbing layer well suited for absorbingphotonic energy. For example, the interior-facing surfaces of lateralwalls defining reaction chamber 104 can be at least partially covered bya light absorbing layer. In some embodiments, additional energy sources204 could be focused on these additional light absorbing layers tofurther increase the rate at which energy can be injected into reactionchamber 104. In some embodiments, the energy sources 204-1 and 204-2 maybe adjustable such that they emit different energy levels or areotherwise adjustable (e.g., with one of the energy sources being closerto the reaction chamber 104 than the other) such that the lightabsorbing layers 106-1 and 106-2 receive different amounts of energy. Insome embodiments, alternatively or additionally, the light absorbinglayers 106-1 and 106-2 may be different (e.g., in composition, indimensions such as thickness or surface area) such that they areconfigured to absorb different amounts of energy.

FIG. 3A shows an example configuration where light absorbing layers106-1 and 106-2 can be positioned upon or adjacent to opposinginterior-facing surfaces of housing 202, with optical elements 302-1 and302-2 that may be used to alter the characteristics of light generatedby energy sources 204-1 and 204-2. In some embodiments, theseinterior-facing surfaces of housing 202 may define reaction chamber 104and the light absorbing layers 106 may be positioned upon (e.g., formedon, deposited on, adhered to, or otherwise disposed on) theinterior-facing surfaces such that the lighted absorbing layers 106 maycome into direct contact with a solution within reaction chamber 104.Alternatively, a substrate may be disposed over one or more of the lightabsorbing layers 106-1 and 106-2, such that they do not come into directcontact with the solution. FIG. 3A also shows how energy sources 204-1and 204-2 can include one or more optical elements 302-1 and 302-2configured to concentrate the light generated by energy sources 204-1and 204-2 onto absorbing layers 106-1 and 106-2. Optical elements 302-1and 302-2 can take the form of one or more lenses, light pipes, orbaffles that at least partially collimate the light generated by energysources 204-1 and 204-2. For example, optical elements 302-1 and 302-2may include convex lenses that serve to focus the light generated bytheir respective energy sources. As another example, optical elements302-1 and 302-2 may include baffles (e.g., with reflector elements) thatare configured to focus the light generated by their respective energysources. Optical elements 302-1 and 302-2 may help reduce the occurrenceof waste light (e.g., by focusing light emitted by energy sources 204-1and 204-2 such that almost all the light is incident on the lightabsorbing layers 106-1 and 106-2) and maximize the amount of poweravailable to add into a solution within reaction chamber 104. In someembodiments, other optical elements can be used to help guide uniformlight from energy sources 204-1 and 204-2 toward reaction chamber 104.For example, a light pipe could be used to transport light from energysources 204-1 and 204-2 directly to various locations on reactionchamber 104. The light pipe could be advantageously shaped to deliverlarger amounts of light to specific regions of light absorbing layers106-1 and 106-2. In some embodiments, the light pipe could extend atleast partially within housing 202. In some embodiments, as describedabove, the energy sources 204-1 and 204-2 may be adjustable, the lightabsorbing layers 106-1 and 106-2 may be different, and/or the opticalelements 302-1 and 302-2 may be different, such that absorption of lightby the light absorbing layers 106-1 and 106-2 may be fine-tuned.

FIG. 3B shows how multiple reaction chambers 104-1, 104-2, and 104-3 canbe positioned within a housing 304 of a reaction vessel 300. In someembodiments, solution can be transferred between adjacent reactionchambers 104-1, 104-2, and 104-3 by internal channels that can bedefined by housing 304. In this way, one stream of solution can traversemultiple reaction chambers 104-1, 104-2, and 104-3. Alternatively, thereaction chambers 104-1, 104-2, and 104-3 may not be separated byphysical barriers, and as such may not require channels. For example,the reaction chambers 104-1, 104-2, and 104-3 illustrated in FIG. 3B maybe disposed within a single chamber of the reaction vessel 300 with nopartitions in between. In some embodiments, as illustrated in FIG. 3B,the reaction chambers may have light absorbing layers made up of severaldiscrete regions (e.g., 106-1 a, 106-1 b, and 106-1 c, 106-2 a, 106-2 b,and 106-2 c). In some embodiments, these discrete regions may beseparated from each other. In some embodiments, any of these discreteregions may have different characteristics, such that they havedifferent temperature profiles. For example, discrete regions 106-1 aand 106-2 a could have a different thickness and/or composition thandiscrete regions 106-1 b and 106-2 b, allowing for energy to be absorbedmore quickly into a solution within the reaction chamber 104-1 ascompared to the reaction chamber 104-2. Alternatively or additionally,energy absorption among different reaction chambers 104 may be varied byadjusting the power levels of the energy sources 302. Varying energyabsorption among reaction chambers 104-1, 104-2, and 104-3 may bebeneficial where multi-step reactions, one or more of which requiredifferent temperatures, are necessary. For example, a single PCR cyclehas multiple steps that require different temperatures (e.g., fordenaturing DNA, annealing, and extending).

In some embodiments, temperatures within reaction chambers 104-1, 104-2,and 104-3 may be varied by assigning multiple energy sources to eachreaction chamber 104. This may be particularly advantageous in caseswhere energy sources 204 may only have ON and OFF states. For example,three energy sources 204 may be assigned to a single reaction chamber104-1. One or more of these energy sources 204 may be turned ON, and thenumber of energy sources 204 that are ON would determine the amount ofenergy delivered to the reaction chamber 104-1. By turning ON and OFFindividual energy sources 204, the temperature within the reactionchamber 104 may be varied along a scale. In this example, turning ON all3 energy sources 204 may result in the highest temperature value,turning on two of the energy sources 204 may result in an intermediatetemperature value, and turning on one of the energy sources 204 mayresult in a low temperature value. In some embodiments, as illustratedin FIG. 3B, the effects of energy sources 204 may not be limited to asingle reaction chamber (e.g., the reaction chamber 104-1). For example,the left-most energy source illustrated in FIG. 3B may deliver energy toboth 104-1 and 104-2. As such, energy received by any one of thechambers 104-1, 104-2, and 104-3 may be varied (e.g., across a gradient)by turning ON or OFF the illustrated energy sources 204. In someembodiments, energy sources 204 could also be equipped with controllersthat allow for a variety of duty cycles to be applied that effectivelyallow the amount of light emitted by each of energy sources 204 to varygreatly. For example, a pulsed control signal could be provided to oneof energy sources 204 that results in that energy source 204 onlytransmitting light 50% of the time, thereby effectively halving theamount of light transmitted to a light absorbing layer as compared to adifferent energy source 204 that transmits light 100% of the time.

FIG. 4 shows an example embodiment in which an energy source takes theform of a chip on board (COB) LED 402 that may be well suited fordistributing light evenly across a relatively large area of housing 304of reaction vessel 300. In some embodiments, COB LED 402 can beconstructed from multiple LED chips that are surface mounted to aprinted circuit board (PCB). In these embodiments, each of the LED chips(or subsets thereof) may be individually controlled such that an amountof light energy outputted and/or a direction at which the light energyis outputted may be controlled (similar to FIG. 3B). One advantage ofthe COB LED is that reaction chambers 104 can be separated by anyinterval or pattern without the need for rearranging individual energysources. This type of configuration also reduces the need for opticalelements that target specific reaction chambers 104. In someembodiments, however, COB LEDs 402 and 404 could include baffles oroptical elements that are configured to prevent the light emitted fromCOB LEDs 402 and 404 from being diverted to either side of housing 304.

FIG. 5A shows an example embodiment with a PCB 502 supporting multipleLEDs 504 that can be used to illuminate opposing sides of a reactionchamber 104 of reaction vessel 506. The light emitted by LEDs 504 can betransmitted by optical fibers 508, which are able to receive andtransmit that light with little or no loss to a point just above anexterior surface of housing 510 of reaction vessel 506. Optical fibers508 may include receiving ends 507 and transmitting ends 509 that areconfigured to efficiently gather and transmit light, respectively.Although FIG. 5A illustrates a PCB supporting two LEDs that illuminateopposing sides of a reaction chamber, the disclosure contemplates a PCBsupporting any number of LEDs that illuminate any number of suitableregions of a reaction vessel.

FIG. 5B shows an example embodiment where light energy from a singleprecursor energy source (e.g., the LED 204) is split between two opticalfibers 508 to illuminate opposing sides of reaction chamber 104 ofreaction vessel 506. In the illustrated example, the LED 204 is housedwithin an LED housing 505 to which receiving ends 507 of the opticalfibers 508 are coupled. The LED housing 505 may be optimized withoptical elements and/or reflectors (e.g., disposed along itsinterior-facing surfaces) for maximizing efficiency of lighttransmission. Although FIG. 5B illustrates splitting light energy from aone LED between two optical fibers to illuminate opposing sides of areaction chamber, the disclosure contemplates splitting light energyfrom any number of LEDs among any number of optical fibers to illuminateany number of suitable regions of a reaction vessel.

FIG. 5C shows the energy source configuration depicted in FIG. 5A withthe addition of an excitation light source assembly and an emissiondetecting sensor assembly. In some embodiments, the excitation lightsource assembly can include excitation LED 512. In some embodiments,excitation LED 512 can be configured to emit visible-spectrum light thatis able to cause any fluorescent markers present within reaction chamber104 (e.g., fluorescent markers bound to DNA strands or nucleotides) toemit fluorescent light. In some embodiments, an excitation filter 514may optionally be used to remove any wavelengths of light generated byexcitation LED 512 that are outside of a wavelength range that excitesthe fluorescent markers. In some embodiments, a wavelength range ofexcitation filter 514 can be adjusted to match fluorescent markers beingused for a particular experiment or reaction. In some embodiments, theemission detecting sensor assembly can include emission detecting sensor515 configured to detect a fluorescent light emitted by fluorescentmarkers within the reaction chamber in response to light from theexcitation LED 512. In some embodiments, an emission filter 516 mayoptionally be used to optimize (e.g., amplify, filter) light that istransmitted to the emission detecting sensor 515. In some embodiments,emission detecting sensor 515 can take the form of a photodiode, CMOS orCCD sensor capable of receiving emissions from the fluorescent markerswithin reaction chamber 510. In some of these embodiments, thephotodiode, CMOS or CCD sensors can be calibrated so that in addition torecognizing the presence of the fluorescent markers, a position of thefluorescent markers within reaction chamber 104 can also be determined.It should be noted that while the emission detecting sensor assembly isdepicted as being on an opposite side of reaction chamber 104 from theexcitation light source assembly that in some embodiments, the twoassemblies can be positioned in any suitable location with respect toeach other (e.g., on the same side of reaction chamber 104). Forexample, the excitation light source assembly could be positioned on thesame side, but offset laterally by about 30 degrees. In someembodiments, the emission filter 516 (which may allow only certainwavelengths to pass through) may serve to block or filter out lightemitted by the optical fibers 508 such that the light emitted by theoptical fibers 508 does not intermix with the light from the excitationassembly (e.g., the excitation LED 512) so that sensor readings of theemission detecting sensor 515 are based on fluorescent light and notcompromised by light from the optical fibers 508. For example, theemission filter may be configured to allow light of one or morewavelengths corresponding to the florescent light, and block or filterout light of one or more other wavelengths (e.g., thereby filtering outor at least significantly reducing light from the optical fibers 508).Additionally, at least in some embodiments, all or most of the lightfrom the optical fibers 508 used to illuminate light absorbing layers106 may generally be prevented from entering into reaction chamber 104by the light absorbing layers 106. As such, intermixing may not be asmuch of an issue in these embodiments. In some embodiments, a systemassociated with the reaction vessel may log (e.g., within a memory) oneor more of an indication that a threshold amount of fluorescent lightwas detected (e.g., an amount above background artifact signals from afluorescent dye), a value quantifying the amount of emitted fluorescentlight, a position within a reaction vessel or chamber at which thefluorescent light was detected, a time point at which the florescentlight was detected, and a temperature of the associated reaction chamberat the time point at which the florescent light was detected. In theseembodiments, the logged data can be used to synthesize information abouta reaction, assay, or experiment. For example, a graph illustrating atemperature and fluorescence profile during an assay or reaction (e.g.,PCR) may be mapped for analysis.

FIGS. 6A-6B shows experimental data from three consecutive heating andcooling test cycles comparing temperature profiles of a reaction chamberbeing heated from one side (one-sided heating) and a reaction chamberbeing heated from two sides (dual-sided heating). Contrary to earlypredictions, dual-sided heating does not simply result in a reactionchamber being heated at twice the speed as one-sided heating. In fact,unpredictably, the ramp-up times with dual-sided heating occur at muchmore than twice the speed of ramp-up times with one-sided heating. Forexample, as illustrated in the graph of FIG. 6A and the correspondingtable of FIG. 6B, it took 6.81 seconds for the reaction chamber to beheated from room temperature to about 95° C. (i.e., the first testcycle) using dual-sided heating, while it took 27.43 seconds for thesame temperature increase using one-sided heating. As such, double-sidedheating in this cycle was about four times faster than one-sidedheating. Similarly, ramp-up times in the second and third test cycleswere more than four times faster in the case of double-sided heating, asshown in FIGS. 6A-6B. Even more unpredictably, the cool-down times(e.g., during which energy was not transmitted to light absorbing layersof the reaction chamber to cause a temperature decrease in the reactionchamber) was also affected. For example, referring to the first testcycle in FIGS. 6A-6B, it took 4.71 seconds for the reaction chamber toramp down from about 95° C. to about 65° C. in a reaction chamber thatwas heated up using double-sided heating, while it took 7.47 seconds forthe same temperature decrease in a reaction chamber that was heated upusing one-sided heating. As such, cooling a reaction chamber heated withdouble-sided heating was about 1.6 times faster in this cycle. Cool-downtimes in the other cycles were similarly faster with a reaction chamberthat was heated with double-sided heating. These unpredictable resultsare at least in part due to the fact that, with dual-sided heating, lessheat is dissipated from the light absorbing layers of reaction chambersto the housing surrounding the reaction chamber while getting thereaction chamber up to a desired temperature. For example, heating fromboth sides heats the reaction chamber more quickly from the beginning.The effects of this quicker heating compounds synergistically over aheating period, because less time spent heating translates to less lossof heat to the housing around the reaction chamber (in one-sidedheating, the additional heat loss would need to be compensated forduring the heating period to achieve a desired temperature in thereaction chamber). In addition, introducing heat from opposing sidesacts to confine heat within the reaction chamber, thereby again reducingloss of heat. For example, in one-sided heating, a first side opposite alight absorbing layer disposed on a second (heated) side may allow for atemperature gradient that facilitates heat from the reaction chamber toescape via the first side. By contrast, such a gradient would not existin dual-sided heating, thereby trapping heat within the chamber, andthereby speeding up heating. As for the quicker cool-down times, thistoo is at least in part due to the reduced loss of heat in dual-sidedheating. For example, since less heat is absorbed by the surroundinghousing during dual-sided heating, when energy is no longer transmittedto let absorbing layers of the reaction chamber, more heat can bedissipated away from the reaction chamber to the housing surrounding thereaction chamber. By contrast, in single-sided heating, since thehousing already has a relatively higher temperature due to more heatdissipation, the temperature gradient between the reaction chamber andthe surrounding housing is much smaller. This may result in slower heattransfer away from the reaction chamber, resulting in slower cool-downtimes.

FIG. 7 illustrates an example method 700 for operating atemperature-controlled reaction vessel system. The method may begin atstep 710, where a reagent is introduced into a first reaction chamber,wherein the first reaction chamber comprises a first light absorbinglayer and a second light absorbing layer, the first and second lightabsorbing layers having an inner surface that is oriented toward aninterior of the first reaction chamber and an outer surface that isoriented away from the interior of the first reaction chamber. At step720, a first energy source may direct a first light toward the outersurface of the first light absorbing layer so as to heat the first lightabsorbing layer. At step 730, a second energy source may direct a secondlight toward the outer surface of the second light absorbing layer so asto heat the second light absorbing layer. At step 740, heat from thefirst and second light absorbing layers may be transferred to thereagent.

Particular embodiments may repeat one or more steps of the method ofFIG. 7, where appropriate. Although this disclosure describes andillustrates particular steps of the method of FIG. 7 as occurring in aparticular order, this disclosure contemplates any suitable steps of themethod of FIG. 7 occurring in any suitable order. Moreover, althoughthis disclosure describes and illustrates an example method foroperating a temperature-controlled reaction vessel system, including theparticular steps of the method of FIG. 7, this disclosure contemplatesany suitable method for operating a temperature-controlled reactionvessel system, including any suitable steps, which may include all,some, or none of the steps of the method of FIG. 7, where appropriate.Furthermore, although this disclosure describes and illustratesparticular components, devices, or systems carrying out particular stepsof the method of FIG. 7, this disclosure contemplates any suitablecombination of any suitable components, devices, or systems carrying outany suitable steps of the method of FIG. 7.

FIG. 8A shows an example reaction vessel 800 with a housing 802 thatdefines multiple reaction chambers 104 that can be filled with solutionand a chamber 805 that includes a temperature sensor 158. In someembodiments, a solution (e.g., one containing nucleotides or reagents)can be introduced into reaction chambers 104-1 and 104-2 of reactionvessel 800. As illustrated, reaction vessel 800 may also include chamber805, which includes temperature sensor 158. In some embodiments, thechamber 805 may be filled with a viscous fluid (e.g., a liquid polymer).The viscous fluid may help keep temperature sensor 158 in a fixedposition within reaction chamber 805. In some embodiments, the viscousfluid can take the form of a liquid polymer or adhesive that can becured (and solidified) to further prevent movement of temperature sensor158 within chamber 805. Similar to the reaction chambers 104, thechamber 805 may be associated with one or more light absorbing layersthat are configured to heat the chamber 805 along with the reactionchambers 104. For example, as illustrated in FIG. 8A, the reactionvessel 800 may have a first light absorbing layer that includes discreteregions 106-1 a, 106-1 b, and 106-1 c and a second light absorbing layerthat includes discrete regions 106-2 a, 106-2 b, and 106-2 c. In thisexample, the discrete regions 106-1 a, 106-1 c, 106-2 a, and 106-2 c areassociated with reaction chambers 104-1 and 104-2, and the discreteregions 106-1 b and 106-2 b are associated with the chamber 805. In someembodiments, temperature sensor 158 may be placed within a centralregion of reaction chamber 805 that allows it to gather an averagetemperature of the chamber 805. It should be noted that in someembodiments the average temperature may not be at the absolute center ofreaction chamber 805 and this position may be shifted (e.g., closer toor farther away from the light absorbing layer) to achieve desiredtemperature sensor readings. In some embodiments, each of reactionchambers 104-1, 104-2 and 805 may be similarly sized and may receiveabout the same amount of light from energy source 204. As such, each mayreceive about the same amount of photonic energy and in some instancesresults in a temperature within reaction chambers 104-1, 104-2 and 805being substantially the same. This allows for accurate measurement oftemperature within multiple reaction chambers using only a singletemperature sensor 158. Having a temperature sensor within a chamber maybe particularly advantageous in some cases, particularly where heattransfer rate is extremely quick (as may be the case with the heatingmechanisms involving light absorbing layers as described herein).Conventional temperature monitoring systems, which place temperaturesensors outside the reaction vessel (e.g., within a platform system ontowhich the reaction vessel is mounted), are typically not effective withsuch quick-heating mechanisms and can create issues with over- orunder-heating, due to inaccurate real-time measurements. For example,such systems may include too much material and/or multiple layers (e.g.,of the housing of the reaction vessel and/or of the platform system) forefficient heat transfer between the reaction chamber and the temperaturesensor. As such, there may be a delay in detecting temperature changesaccurately. While this delay may be tolerable in cases where reactionchambers are heated relatively slowly, it can be impermissible in caseswhere heating occurs rapidly. By placing temperature sensors within achamber so as to approximate conditions within reaction chambers,real-time measurements may be accurate.

FIG. 8B shows a reaction vessel 820 that includes a housing 822 definingat least two reaction chambers 104, a chamber 805 that includes atemperature sensor 158, and a light diffusing layer 806. The temperaturesensor 158 may be capable of measuring an internal temperature withinthe chamber 805. In some embodiments, the reaction chambers 104 and thechamber 805 may be configured to have the same dimensions (e.g., thesame size and shape) and may have the discrete regions 106-2 a, 106-2 b,and 106-2 c that are substantially the same. In other embodiments, anyof these characteristics may be varied. In some embodiments, thecontents of reaction chambers 104 may be different than the chamber 805.As described elsewhere, the chamber 805 can be filled with a pottingmaterial (e.g., a viscous fluid or a cured polymeric material) that maybe configured to pre-fill the chamber 805 such that it prevents orreduces movement of sensor 158 within the chamber 805. For example, thechamber 805 may be filled with a polymeric material such as asilane-modified polymer or liquid adhesive that can be converted to asolid by undergoing a thermal- or UV-curing operation.

Filling the chamber 805 with a potting material offers the ability tofix the temperature sensor 158 in place within reaction chamber 104 andtransfer heat from light absorbing material to temperature sensor, sothat temperature measurements are more precise. Using a potting materialrather than a water-based solution that may more closely mimic thesolution in reaction chambers 104 has the added advantage of affordingconvenient reusability. For example, in some embodiments, the reactionvessel 820 may be configured to be reused over the course of a largenumber of different operations. In these embodiments, the chamber 805may be filled with a potting material (e.g., a polymeric material) thathas material properties allowing it to withstand a large number ofthermal cycles. If the chamber 805 were filled with, for example, wateror a water-based solution, the water or water-based solution mayevaporate over time (e.g., especially considering that many reactionswithin the reaction vessel may be conducted at high temperatures) suchthat temperature readings would not be precise over time. Moreover, thechamber 805 cannot be filled with air, because air is not a medium thatallows for efficient heat transfer and therefore does not allow foraccurate temperature measurements. As such, a potting material such as apolymeric material is particularly effective.

While the potting material can also be chosen to match a thermalconductivity and specific heat of the solution in the other reactionchambers 104, in some cases, an exact match may not be possible. Forexample, a specific heat of a polymeric material in chamber 805 can besubstantially lower than the contents of reaction chambers 104 (e.g., awater-based PCR solution with DNA, polymerase enzyme), such that thetemperature profile of the chamber 805 may be different from thetemperature profile of the reaction chambers 104. As a result, in thisexample, the polymeric material in chamber 805 may heat more quicklythan the water-based solution in the other reaction chambers 104, eventhough the light sources 204 a, 204 b, and 204 c may be directing thesame amount of energy toward the reaction chambers 104 and the chamber805. As such, in this example, the temperature value detected by thetemperature sensor 158 may not be an accurate representation of thetemperature within the reaction chambers 104. To rectify this differencein the rate of heating, a light diffusing layer 806 can be added to thereaction vessel in between energy sources and the chamber 805. Forexample, as illustrated in FIG. 8B, the light diffusing layer 806 may bedisposed along a surface of housing 822, such that light energy from theenergy sources (e.g., the energy source 204 b) must travel through thelight diffusing layer 806 before it hits the discrete region 106-2 b. Insome embodiments, a light diffusing layer may be a material that isconfigured to reflect or scatter a portion of the light that is incidentto the light diffusing layer. In some embodiments, light diffusing layer806 can be specifically tuned to reduce the amount of light incident tolight absorbing layer 106-2 b by an amount calculated to account for adifference between the specific heat of the potting material in thechamber 805 and the (e.g., water-based) solution within the otherreaction vessels 104. This configuration reduces the amount of lightreceived at the light absorbing layer 106-2 b, which can help compensatefor the lower specific heat of the potting material in the chamber 805.In some other embodiments, a light blocking layer may be used in placeof a light diffusing layer. For example, a light blocking layer mayinclude one or more opaque sections that prevent segments of lighttransmitted by an energy source from passing through toward the chamber805. In other embodiments, a filter layer may be employed to filter outcertain wavelengths of light, such that the light that passes the filterlayer is of reduced energy. In a configuration such as the one shown inFIG. 8B, an output of the energy source 204 b directed at discreteregion 106-2 b can also be reduced to help compensate for the thermalcharacteristics of the potting material within the chamber 805. Althoughthis disclosure focuses on a potting material within chamber 805 thathas a lower specific heat than the contents of reaction chambers 104,this disclosure also contemplates embodiments with a potting materialwithin chamber 805 that has a higher specific heat than the contents ofreaction chambers 104. In these embodiments, the reaction vessel may bevaried to increase absorption of heat energy of chamber 805 so as tocompensate for the difference.

FIG. 8C shows a reaction vessel 850 having a housing 854 defining achamber 805 that is different from the reaction chambers 104. Asillustrated, chamber 805 includes a temperature sensor 158 that issurrounded and fixed in place (e.g., by a solidified polymericmaterial). The reaction chambers 104 can be filled, for example, withsolution that includes various chemicals and/or biological materials.The reaction chambers 104 may include discrete regions 106-2 a and 106-2c of a light absorbing layer for converting energy from photons emittedby energy source 808 into heat energy that is then used to distributeheat by thermal conduction and/or convection within reaction chambers104. The chamber 805 may include discrete region 106-2 b fordistributing heat to the chamber 805. As depicted, energy source 808 cantake the form of a chip on board (COB) LED that may be well suited fordistributing light evenly across a large area.

FIG. 8C also shows how in embodiments where a specific heat of thepotting material is different than the specific heat of the solutionwithin the other reaction chambers 104, the dimensions of chamber 805may be varied. For example, in the case where a polymeric material witha lower specific heat is used to fill chamber 805, a size of the chamber805 may be increased. Increasing the size of chamber 805 may increasethe amount of the area and the potting material therein that has to beheated by the heat energy released from the discrete region 106-2 b,thereby slowing a rate at which the chamber 805 increases intemperature. Increasing the dimensions of chamber 805 also increases thedistance heat has to travel to arrive at temperature sensor 158, and maythereby further reduce a detected rate of temperature increase. In someembodiments and as depicted, the dimensions or composition of thediscrete region 106-2 b may be varied such that the discrete region106-2 b absorbs less energy than the discrete regions 106-2 a and 106-2c. For example, the discrete region 106-2 b may have a reduced thickness(e.g., 100 nm) when compared to the discrete regions 106-2 a and 106-2 c(e.g., 200 nm). In this example, the volume of material making up thediscrete region 106-2 b is reduced, which may in turn reduce the amountof heat that can be stored and thermally conducted into the chamber 805.As a result, the rate at which energy is able to be introduced into thechamber 805 is reduced, thereby compensating for the characteristics ofa potting material of chamber 805 with a lower specific heat thanreaction chambers 104. As another example, the discrete region 106-2 bmay be composed of a material that absorbs light energy at a reducedrate as compared to the discrete regions 106-2 a and 106-2 c. In someembodiments, a position of temperature sensor 158 can be varied tocompensate for temperature profile differences. For example, thetemperature sensor 158 may be biased away from discrete region 106-2 bto reduce the heating rate measured by temperature sensor 158.

FIG. 8D shows an example reaction vessel 870 having a light diffusionlayer 806 and a light reflecting layer 810. The reaction vessel 870 mayinclude a housing 874 defining multiple reaction chambers 104 and achamber 805 including a potting material (e.g., a solidified polymericmaterial) and a temperature sensor 158 (e.g., surrounded by and fixed inplace by the solidified polymeric material). The reaction chambers 104can be filled with solution that may include various chemicals and/orbiological materials. The reaction vessel 870 may include a lightabsorbing layer that includes multiple discrete regions 106-2 a, 106-2b, and 106-2 c for converting energy from photons emitted by energysource 808 into heat energy that is then distributed by thermalconduction and/or convection within reaction chambers 104. Asillustrated in FIG. 8D, a position of temperature sensor 158 can bebiased away from discrete region 106-2 b to reduce a heating ratemeasured by temperature sensor 158.

FIG. 8D shows light diffusing layer 806, which as discussed above can betuned to adjust an amount of light that reaches the discrete region106-2 b. In the illustrated example, the light diffusing layer 806reduces the amount of light that is able to enter into housing component874 and illuminate the discrete region 106-2 b. One additional way toadjust an amount of light that reaches the discrete region 106-2 b is to(alternatively or additionally) include a light reflecting layer 810that blocks all light from passing through a region of housing 874. Insome embodiments, as depicted, light reflecting layer may be positioneddirectly beneath temperature sensor 158, which can further reduce theamount of heat that reaches temperature sensor 158. Other configurationsof light reflecting layer are also possible including a checkerboardpattern or a striped pattern that more evenly reduces the amount oflight able to enter and pass through light diffusing layer 806. In someembodiments, light diffusing layer 806 and light reflecting layer 810can be incorporated into a single layer. It should be noted that any ofthe depicted reaction vessels configurations can include any combinationof the energy attenuating features (e.g., light diffusion layers, lightreflection layers, light blocking layers, filters) illustrated in any ofFIGS. 8B-8D and described in the related descriptions. Although thedepicted reaction vessels display the energy attenuation features beingexternal to the housing (e.g., referencing FIG. 8D, the light diffusionlayer 806 and the light reflecting layer 810 are exterior to housing874), the energy attenuation features may be disposed in any suitablelocation, including within the housing (e.g., referencing FIG. 8D, at alocation within housing 874 in between the discrete region 106-2 b andthe energy source 808).

FIG. 8E shows an embodiment in which a temperature sensor can beembedded within a housing 884 of a reaction vessel 880. Temperaturesensors can be embedded in a variety of different positions outside achamber. For example, referencing FIG. 8E, a temperature sensor may beembedded within housing 884 at positions 159-1 and/or 159-2, asdepicted. In some embodiments, a temperature sensor can be embedded atposition 159-2 by adhering light absorbing layer directly atop thetemperature sensor. Embedding a temperature sensor in any of thesepositions may allow all the depicted reaction chambers 104 to conductnormal operations since temperature sensor does not need to be withinits own chamber (e.g., the chamber 805 depicted in FIGS. 8A-8D) that isfilled with a potting material such as a polymeric material. Sincetemperature sensor 158 is not in direct contact with solution within anyof reaction chambers 104, a calibration function can be applied to itsreadings so that temperature sensor is able to provide accuratetemperature readings of solution within one of reaction chambers 104over a predefined range of operating temperatures. In some embodiments,temperature sensors may be embedded within housing 884 by boring a holethrough the housing 884, inserting the temperature sensor and associatedcircuitry (e.g., a conducting wire), and sealing the hole. In otherembodiments, the temperature sensors may be molded directly into thehousing 884.

FIG. 8F shows an example embodiment in which temperature sensor 158 isdisposed within a module 890 that is separate and distinct from ahousing 884 of a reaction vessel 880. As illustrated, the temperaturesensor 158 may be disposed within a chamber 805 of the module 890.Chamber 805 is defined by walls of a housing 892 of module 890 and canbe filled with potting material as described above. In this way, vessel890 can take the form of a separate temperature-sensing assembly. Havingthe temperature sensing assembly as its own module allows for thetemperature-sensing assembly to be maneuvered into different positionsrelative to reaction vessel 880 as needed to achieve proper calibrationof temperature sensor 158. For example, module 890 could be positionedcloser to or farther from energy source 808 to account for differencesin specific heat between the potting material surrounding temperaturesensor 158 and the solution within the reaction vessels 104. Having aseparate temperature-sensing assembly also allows for thetemperature-sensing assembly to be replaced without having to replace ortroubleshoot a temperature sensor incorporated within the much largerreaction vessel 880. As such, this modular separation may serve toreduce costs and provide for more convenient fixes to issues related totemperature monitoring. In some embodiments, reaction vessel 880 andmodule 890 can be coupled together (e.g., directly secured together byan attachment mechanism) to achieve a well-defined distance (e.g., asdefined by an attachment mechanism) between the reaction vessels andenergy source 808. In the example embodiment illustrated in FIG. 8F, theenergy source 808 may direct energy toward both the reaction vessel 880and the module 890. As such, when the energy source 808 directs energytoward the reaction vessel 880 and the module 890, the discrete region106-2 b of the module 890 and the discrete regions 106-2 a, 106-2 d, and106-2 c of the reaction vessel 880 may absorb the energy and therebyheat the reaction chambers 104 and the chamber 805. In some embodiments,multiple energy sources (e.g., one or more LEDS for the reaction vessel880 and a separate LED for the module 890) may be used.

Although FIGS. 8B-8F illustrates example reaction vessels that includeonly one light absorbing layer, this disclosure contemplates any numberof light absorbing layers (e.g., two light absorbing layers asillustrated in FIG. 8A). Additionally, any suitable number of lightsources may be used (e.g., on opposing sides of the reaction vessel).Moreover, although FIGS. 8A-8F illustrate example reaction vessels witha light absorbing layer that includes multiple discrete regions, witheach discrete region corresponding to one chamber, this disclosurecontemplates that a single light absorbing layer may correspond tomultiple chambers and further contemplates that multiple discreteregions may correspond to a single chamber.

FIG. 9A shows a lateral schematic view of a cross-section of an examplereaction vessel 980 with a chamber 805 containing a temperature sensor158. In some embodiments, the temperature sensor 158 may be athermocouple, a thermistor, a resistance temperature detector (RTD), orany other suitable sensor that may be used to determine temperature. Theillustrated chamber 805 may be filled with a potting material (e.g., acured polymeric material) as described elsewhere herein. In someembodiments, the reaction vessel 980 may be formed by forming andaffixing various components of the reaction vessel 980 together. In someembodiments, housing 900 may be formed by, for example a process ofinjection molding. For example, a top portion 900-1 and a bottom portion900-2 of housing 900 may be separately injection molded. In thisexample, both portions may have depressions that are to define reactionchambers (e.g., reaction chambers 104, which are not visible in FIG. 9A)and/or temperature-monitoring chambers (e.g., chamber 805) when theportions are secured together. Alternatively, only a first portion mayhave depressions, in which case the second portion may simply overlaythe depressions of the first portion to create chambers. Lightabsorption layers (e.g., including discrete regions 106-1 and 106-2) maybe adhered or plated onto the gaps such that when the top portion 900-1and bottom portion 900-2 are secured together, the light absorptionlayers are long interior-facing surfaces of the resulting chambers(e.g., chamber 805 as illustrated). A temperature sensor 158 may bepositioned within the chamber 805 in a desired position. In someembodiments, a polymeric material (or any other suitable pottingmaterial) may be injected into chamber 805. While the temperature sensor158 is in the desired position, the polymeric material may be cured andsolidified, such that the temperature sensor 158 is fixed in the desiredposition. Top portion 900-1 and bottom portion 900-2 may be securedtogether by any suitable means (e.g., screws, adhesives, snap-fitting).In some embodiments, as illustrated in FIG. 9A, energy attenuatingfeatures such as light diffusion layers 806-1 and 806-2 may be adheredor otherwise affixed to the housing 900 adjacent to chambers (e.g.,chamber 805) with temperature sensors (e.g., temperature sensor 158).

FIG. 9B shows an overhead schematic view of a cross-section of theexample reaction vessel 980 depicted in FIG. 9A with a chamber 805having a temperature sensor 158 coupled to a processor 910. Asillustrated, the temperature sensor 158 may be coupled to a processor910 that may be configured to, among other things, determine atemperature value based on a temperature signal attracted by thetemperature sensor 158. In some embodiments, a physical connection maybe present between the temperature sensor 158 and the processor 910. Inother embodiments, a wireless connection may be used to transmit atemperature signal from the temperature sensor 158 to the processor 910.FIG. 9B also illustrates that the reaction vessel 980 may include nearthe chamber 805 a reaction chamber 104, whose internal temperature maybe approximated by the internal temperature of the chamber 805. In someembodiments, as illustrated in FIG. 9B, the processor 910 may beexternal and separate from the housing 900 of the reaction vessel. Inother embodiments, the processor 910 may be within the housing 900 oraffixed to the housing 900.

FIG. 10 shows a simplified schematic diagram of a reaction vessel system1000. As illustrated, a PCB 1010 (e.g., which may include the processor910 referenced in FIG. 9B) may interface with various elements of thereaction vessel system 1000. For example, the PCB 1010 may interfacewith an LED driver 1020, which may drive one or more LEDs. In theexample system illustrated in FIG. 10, the LED driver 1020 drives LEDs1025-1 and 1025-2. In this example, the LED 1025-1 is configured todirect light at a chamber 805 comprising a temperature sensor 158, andthe LED 1025-2 is configured to direct light at a reaction chamber 104.The chamber 805 and the reaction table 104 may be within a housing of areaction vessel or may be separated (e.g., the chamber 805 may be in aseparate module). One or more reactions may be made to occur within thereaction chamber 104, and temperatures within the reaction chamber 104may be regulated by adjusting the LED 1025-2. The LED 1025-1 may beadjusted along with the LED 1025-2 so that a measured temperature withthe chamber 805 may be used to estimate temperature within the reactionchamber 104. The temperature sensor 158 within the chamber 805 may becoupled to the PCB 1010 (e.g., to the processor 910 described withrespect to FIG. 9B, which may be within the PCB 1010). In someembodiments, as illustrated in FIG. 10, an amplifier 1040 or any othersuitable circuitry may be disposed in between so as to appropriatelymodulate the signal from the temperature sensor 158. In someembodiments, as illustrated in FIG. 10, an LED driver 1030 may be usedto drive an excitation LED 1035, which may be configured to emit anexcitation light configured to cause any fluorescent markers (e.g.,which may be bound to target molecules, and may thus be used as anindicator for determining the presence of the target molecules withinthe chamber 104 at a given time) within the reaction chamber 104 to emita fluorescent light. The emitted florescent light may be detected by aphotodiode 1028 (or any other suitable emission sensor). In someembodiments, as illustrated in FIG. 10, one or more optics (e.g., theoptics 1027 and 1037) may be used to filter or otherwise modify lightsignals as described above. Circuitry such as signal monitor 1029 may beused to process signals from the photodiode 1028, and may transmit theresulting signal to the PCB 1010. In some embodiments, a processorwithin the PCB 1010 may further process the various signals it receives(e.g., from the signal monitor 1029 and the amplifier 1040) to determineoutputs that may be sent to a user interface/user experience device 1060(e.g., a display device). For example, the user interface/userexperience device 1060 may receive instructions to display one or moreof a temperature detected at the temperature sensor 158, an estimatedtemperature value of the reaction chamber 104 based on the temperaturedetected at the temperature sensor 158 (e.g., by adjusting the detectedtemperature using calibrated functions as described below), anindication of the presence and/or location of an emitted florescentsignal, and any other suitable parameters or values. In someembodiments, the PCB 1010 may drive a feedback loop that is able tomonitor and regulate temperatures within the reaction vessel. Forexample, the PCB 1010 may continuously or semi-continuously receivetemperature information from the temperature sensor 158 and may adjustthe LEDs 1025-1 and 1025-2 to keep temperatures within a desiredtemperature range. In some embodiments, the PCB may also operate a fan1050 (or other suitable cooling device), which may be used to, forexample, help cool down the reaction vessel during cool-down periods(e.g., for a step of a reaction or assay that requires a lowertemperature). Although FIG. 10 illustrates some elements as beingseparate from the PCB 1010, this disclosure contemplates that one ormore of these elements may be part of the PCB 1010. For example, the LEDdrivers 1020 and 1030, the signal monitor 1029, and the amplifier 1040may reside on the PCB 1010. Although FIG. 10 illustrates particularnumbers of temperature-monitoring chambers (the chamber 805), andreaction chamber (the reaction chamber 104), LEDs, emission detectors,etc., this disclosure contemplates any number of such elements (e.g.,multiple chambers 805 for temperature monitoring, multiple reactionchambers 104, multiple LEDs.

FIG. 11A shows experimental data from tests conducted on apolymer-filled chamber and a water-filled chamber (approximatingconditions of a typical reaction chamber, e.g., a water-based solutionwith DNA). As described above, the specific heat of the potting material(e.g., a polymeric material) within a temperature-monitoring chamber(e.g., a chamber 805, as illustrated in FIGS. 8A-8D and 8F) may not havea temperature profile that is identical to a solution within a reactionchamber (e.g., a reaction chamber 104, as illustrated in FIGS. 8A-8D and8F). As can be seen in FIG. 11A, the temperature profiles of thepolymer-filled chamber and the water-filled chamber generally track oneanother, but are slightly different. For example, the polymer that wastested has a lower specific heat than water, which results in thepolymer-filled chamber being heated more quickly than the water-filledchamber (and cooled more quickly than the water-filled chamber). Asmentioned above, a system may compensate for this temperature-profiledifference by adjusting the amount of energy that is transmitted toand/or absorbed by the temperature-monitoring chamber (e.g., by varyingthe compositions or dimensions of the chamber or the correspondingdiscrete region of the light absorbing layer, by varying the lightdirected at the chamber, by adding energy attenuating features).

Additionally or alternatively, the system may compensate for thetemperature-profile difference by calibrating the system accordingly.For example, experiments such as the one that yielded the data reflectedin FIG. 11A may be performed to determine differences between atemperature-monitoring chamber and a reaction chamber. The system maythen be calibrated to account for these differences. For example, thesystem may determine a function for converting a temperature valuedetected in the temperature-monitoring chamber to estimated temperaturevalues for particular reaction chambers. In some embodiments, one ormore lookup tables may be constructed for easy conversion. For example,it may be determined that a temperature of 100° C. in a particulartemperature-monitoring chamber corresponds to a temperature of 97° C. ina particular reaction chamber.

FIG. 11B shows experimental data from 24 successive PCR for 45 cyclesusing a polymer-filled reference chamber as a temperature-monitoringchamber with reference to which the different steps of PCR cycles wereperformed for all 24 successive PCR. In this experiment, temperatureinformation collected from a temperature sensor within a polymer-filledchamber was used to determine when and how long to heat surroundingreaction chambers for the various PCR steps during each cycle.Twenty-four successive PCR were performed in the reaction chambers usingthis methodology. As reflected in FIG. 11B, the time it took to gothrough 45 cycles for each successive PCR remained relativelyconsistent, with a standard deviation of only +/−1.08 seconds. Thisconsistency is evidence of the reliability of repeatedly using apolymer-filled chamber for monitoring temperature for a large number ofsuccessive PCR cycles. In contrast, using a water-filled chamber (wherea temperature sensor was disposed within a chamber containing awater-based solution) as a temperature-monitoring chamber did notprovide the same consistent and reliable results. Performing PCR cyclesbased on temperature information based on the water-filled chamberresulted in significant changes in PCR thermal cycling time withrepeated PCR. This was at least in part because the temperature for aninitial enzyme activation and denaturation temperature (e.g., which maybe generally higher than 95° C.) is high enough to generate the airbubbles within the chamber, which caused inaccurate measurements oftemperature. As a result, relying on such temperature measurements wouldresult in heating and cooling steps being performed for inconsistent,non-optimal periods of time. For example, at a given time point, atemperature sensor within a water-filled temperature-monitoring chambermay incorrectly register a temperature that is below the temperature ofsurrounding reaction chambers. As such, based on this incorrectinformation, the reaction chambers may be heated for longer thannecessary. As successive PCR were performed in experiments where awater-filled temperature-monitoring chamber is used as a reference, themeasured temperatures would vary significantly, resulting ininconsistent times for each PCR. As shown in FIG. 11B, this is not thecase with polymer-filled temperature-monitoring chambers, where each ofthe 24 PCR took approximately 513.35 seconds, with a standard deviationof only 1.08 seconds.

FIGS. 11C-11D show experimental data from PCR performed using apolymer-filled temperature monitoring chamber as a reference, withdifferent initial DNA template concentrations. FIGS. 11C-11D showsdetected fluorescence levels as a function of a number of PCR cycles.The experiment was performed with different initial DNA templateconcentrations. FIG. 11D illustrates the same data as FIG. 11C with thefluorescence values charted along a logarithmic scale. As can be seen inFIGS. 11C-11D, the experiment was conducted with initial DNA templateconcentrations of 10¹, 10², 10³, 10⁴, 10⁵, and a no template control(NTC). The NTC was merely performed as a control to prove that noincrease of a fluorescence signal (as compared to background artifactsignals from a fluorescent dye) would be detected without PCRamplification, and this was in the fact the case—no increase offluorescence signal was detected for this control, as illustrated inFIGS. 11C-11D. In the experiment, PCR were performed for each initialDNA template concentration three times, as illustrated in FIGS. 11C-11D.The average of different data points such as the threshold cycle valuesof the different initial DNA template concentrations were comparedagainst similar data points for conventional PCR methods, and the datawas found to consistently match up. The graphs illustrated in FIGS.11C-11D are generally similar to what the literature shows forconventional PCR with the different initial DNA template concentrationsthat were tested. This illustrates that the high-speed PCR enabled bythe high-speed heating mechanisms of the reaction vessels disclosedherein offer high-quality results. That is, performing the different PCRsteps quickly using the disclosed high-speed mechanisms does not appearto compromise the various PCR steps. The result is a much higherthroughput PCR with equally robust results as the slower conventionalPCR devices.

FIG. 12 shows a block diagram illustrating a method for assembling atemperature monitoring assembly. At 1202, a temperature sensor is placedwithin a reaction chamber of a reaction vessel. In some embodiments, thetemperature sensor can be temporarily held in place by a fixturingdevice that positions the temperature sensor within a central region ofthe reaction chamber. In some embodiments, multiple temperature sensorscan be positioned within a single reaction chamber. This can be helpfulin implementations where a temperature of the reaction chamber isexpected to vary in different regions of the reaction chamber. At 1204,liquid adhesive or polymeric materials are injected into and fill thereaction chamber. In some embodiments, the liquid adhesive can take theform of a silane curing adhesive. At 1206, the liquid adhesive orpolymeric material can undergo a curing operation that solidifies theliquid adhesive or polymeric material. The fixturing device can bereleased from the temperature sensor prior to or during the curingoperation once a position of the temperature sensor with respect to thepolymeric or adhesive material is established. At 1208, the temperaturesensor can be calibrated. Calibration of the temperature sensor can beaccomplished in many ways. For example, light diffusing or lightreflecting layers can be placed between a light absorbing layerassociated with the reaction chamber and an energy source configured toilluminate the light absorbing layer. In some embodiments, calibrationof the temperature sensor can amount to creating a lookup table thatcorrelates readings from the temperature sensor with readings taken bycalibration temperature sensors that are used to monitor a temperatureof solution within adjacent reaction chambers during a calibrationoperation. It should be appreciated that a size of the reaction chamberand position of the temperature sensor within the reaction chamber canalso be adjusted at various points during the assembly operation toachieve a desired thermal profile for the temperature sensor.

Particular embodiments may repeat one or more steps of the method ofFIG. 12, where appropriate. Although this disclosure describes andillustrates particular steps of the method of FIG. 12 as occurring in aparticular order, this disclosure contemplates any suitable steps of themethod of FIG. 12 occurring in any suitable order. Moreover, althoughthis disclosure describes and illustrates an example method forassembling a temperature monitoring assembly, including the particularsteps of the method of FIG. 12, this disclosure contemplates anysuitable method for manufacturing a reaction vessel, including anysuitable steps, which may include all, some, or none of the steps of themethod of FIG. 12, where appropriate. Furthermore, although thisdisclosure describes and illustrates particular components, devices, orsystems carrying out particular steps of the method of FIG. 12, thisdisclosure contemplates any suitable combination of any suitablecomponents, devices, or systems carrying out any suitable steps of themethod of FIG. 12.

FIG. 13 illustrates an example method 1300 for manufacturing a reactionvessel. The method may begin at step 1310, where a first portion of areaction vessel housing may be formed, where the first portion of thereaction vessel housing has a plurality of depressions. At step 1320, asecond portion of the reaction vessel housing may be formed. At step1330, the first and second portions of the reaction vessel housing maybe secured to each other, such that the depressions at least in partdefine a plurality of chambers, the plurality of chambers comprising areaction chamber and a temperature-monitoring chamber. At step 1340, atemperature sensor may be placed at a desired position within thetemperature-monitoring chamber. At step 1350, a liquid material may beintroduced into the temperature-monitoring chamber. At step 1360, theliquid material may be caused to solidify within thetemperature-monitoring chamber.

Particular embodiments may repeat one or more steps of the method ofFIG. 13, where appropriate. Although this disclosure describes andillustrates particular steps of the method of FIG. 13 as occurring in aparticular order, this disclosure contemplates any suitable steps of themethod of FIG. 13 occurring in any suitable order. Moreover, althoughthis disclosure describes and illustrates an example method formanufacturing a reaction vessel, including the particular steps of themethod of FIG. 13, this disclosure contemplates any suitable method formanufacturing a reaction vessel, including any suitable steps, which mayinclude all, some, or none of the steps of the method of FIG. 13, whereappropriate. Furthermore, although this disclosure describes andillustrates particular components, devices, or systems carrying outparticular steps of the method of FIG. 13, this disclosure contemplatesany suitable combination of any suitable components, devices, or systemscarrying out any suitable steps of the method of FIG. 13.

FIG. 14 illustrates an example method 1400 for monitoring a reactionchamber temperature. The method may begin at step 1410, where a firsttemperature over temperature-monitoring chamber associated with areaction vessel is measured. The temperature-monitoring chamber may befilled with a potting material. At step 1420, a signal corresponding tothe first temperature may be transmitted to a processor. At step 1430,the processor may determine a first temperature value corresponding tothe first temperature. At step 1440, a second temperature valueassociated with a reaction chamber may be estimated based on the firsttemperature value.

Particular embodiments may repeat one or more steps of the method ofFIG. 14, where appropriate. Although this disclosure describes andillustrates particular steps of the method of FIG. 14 as occurring in aparticular order, this disclosure contemplates any suitable steps of themethod of FIG. 14 occurring in any suitable order. Moreover, althoughthis disclosure describes and illustrates an example method formonitoring a reaction chamber temperature, including the particularsteps of the method of FIG. 14, this disclosure contemplates anysuitable method for monitoring a reaction chamber temperature, includingany suitable steps, which may include all, some, or none of the steps ofthe method of FIG. 14, where appropriate. Furthermore, although thisdisclosure describes and illustrates particular components, devices, orsystems carrying out particular steps of the method of FIG. 14, thisdisclosure contemplates any suitable combination of any suitablecomponents, devices, or systems carrying out any suitable steps of themethod of FIG. 14.

The foregoing description, for purposes of explanation, used specificnomenclature to provide a thorough understanding of the describedembodiments. However, it will be apparent to one skilled in the art thatthe specific details are not required in order to practice the describedembodiments. Thus, the foregoing descriptions of specific embodimentsare presented for purposes of illustration and description. They are notintended to be exhaustive or to limit the described embodiments to theprecise forms disclosed. It will be apparent to one of ordinary skill inthe art that many modifications and variations are possible in view ofthe above teachings.

1. A reaction vessel system, comprising: a reaction vessel, comprising:a housing having a first interior-facing surface and an opposing secondinterior-facing surface; a first reaction chamber defined by the firstinterior-facing surface and the second interior-facing surface; a firstlight absorbing layer conforming to the first interior-facing surface ofthe housing; and a second light absorbing layer conforming to the secondinterior-facing surface of the housing; a first energy source configuredto direct light through the housing at the first light absorbing layer;and a second energy source configured to direct light through thehousing at the second light absorbing layer.
 2. The reaction vesselsystem of claim 1, wherein the first light absorbing layer and thesecond light absorbing layer each comprise a metallic film formed onrespective first and second interior-facing surfaces of the housing. 3.(canceled)
 4. The reaction vessel system of claim 1, wherein at least aportion of the housing is optically transparent to wavelengths of lightemitted by the first energy source and the second energy source. 5-6.(canceled)
 7. The reaction vessel system of claim 1, further comprisingan excitation light source assembly and an emission detecting sensorassembly, wherein the excitation light source assembly comprises anexcitation light source configured to direct an excitation lightconfigured to cause a fluorescent marker within the first reactionchamber to emit a fluorescent light, and wherein the emission detectingsensor assembly comprises an emission sensor configured to detect theemitted fluorescent light.
 8. The reaction vessel system of claim 7,further comprising an emission filter disposed between the firstreaction chamber and the emission sensor, wherein the emission filter isconfigured to allow light of one or more first wavelengths correspondingto the emitted fluorescent light, and filter out light of one or moresecond wavelengths, wherein the one or more first wavelengths aredifferent from the one or more second wavelengths.
 9. The reactionvessel system of claim 8, further comprising an excitation filterdisposed between the first reaction chamber and the excitation lightsource, wherein the excitation filter is configured to allow light ofone or more third wavelengths configured to excite the fluorescentmarker, and filter out light of one or more fourth wavelengths, whereinthe one or more third wavelengths are different from the one or morefourth wavelengths. 10-11. (canceled)
 12. The reaction vessel system ofclaim 1 further comprising: a second reaction chamber; a third energysource configured to direct light through the housing at the first lightabsorbing layer adjacent to the second reaction chamber; and a fourthenergy source configured to direct light through the housing at thesecond light absorbing layer adjacent to the second reaction chamber;wherein the first energy source, the second energy source, the thirdenergy source, and the fourth energy source are individuallycontrollable.
 13. The reaction vessel system of claim 12, wherein thefirst light absorbing layer comprises a first discrete region associatedwith the first reaction chamber and a second discrete region associatedwith the second reaction chamber, and wherein the first energy source isconfigured to direct light at the first discrete region and the thirdenergy source is configured to direct light at the second discreteregion.
 14. The reaction vessel system of claim 1, wherein the firstenergy source and the second energy source comprise optical fiberscoupled to a single precursor energy source, wherein energy from thesingle precursor energy source is divided between the first energysource and the second energy source.
 15. The reaction vessel system ofclaim 1, wherein the first light absorbing layer and the second lightabsorbing layer having an inner surface that is oriented toward aninterior of the first reaction chamber and an outer surface that isoriented away from the interior of the first reaction chamber, andwherein the first energy source and the second energy source areconfigured to direct light at the outer surfaces of the first lightabsorbing layer and the second light absorbing layer, respectively. 16.The reaction vessel system of claim 1, wherein the housing is configuredto be disposed between the first energy source and the second energysource when in use.
 17. The reaction vessel system of claim 1, whereinthe first light absorbing layer and the second light absorbing layerhave a same thickness and composition.
 18. A method of operating atemperature-controlled reaction vessel system, the method comprising:introducing a reagent into a first reaction chamber, wherein the firstreaction chamber comprises a first light absorbing layer and a secondlight absorbing layer, the first and second light absorbing layershaving an inner surface that is oriented toward an interior of the firstreaction chamber and an outer surface that is oriented away from theinterior of the first reaction chamber; directing, by a first energysource, a first light toward the outer surface of the first lightabsorbing layer so as to heat the first light absorbing layer;directing, by a second energy source, a second light toward the outersurface of the second light absorbing layer so as to heat the secondlight absorbing layer; and causing heat from the first and second lightabsorbing layers to be transferred to the reagent.
 19. The method ofclaim 18, further comprising: introducing a reagent into a secondreaction chamber; causing a third energy source to direct a third lighttoward an outer surface of the first light absorbing layer adjacent to asecond reaction chamber; and causing a fourth energy source to direct afourth light toward an outer surface of the second light absorbing layeradjacent to the second reaction chamber.
 20. The method of claim 19,wherein the first light absorbing layer comprises a first discreteregion associated with the first reaction chamber and a second discreteregion associated with the second reaction chamber, and wherein thefirst light is directed toward the first discrete region and the thirdlight is directed toward the second discrete region.
 21. The method ofclaim 19, wherein the second light absorbing layer comprises a firstdiscrete region associated with the first reaction chamber and a seconddiscrete region associated with the second reaction chamber, and whereinthe second light is directed toward the first discrete region and thefourth light is directed toward the second discrete region.
 22. Themethod of claim 18, further comprising: directing an excitation lightfrom an excitation light source toward the first reaction chamber,wherein the excitation light is configured to cause a fluorescent markerwithin the first reaction chamber to emit a fluorescent light; anddetecting, by an emission detecting sensor assembly, the emittedfluorescent light.
 23. The method of claim 22, further comprising:filtering light reaching the emission detecting sensor assembly with anemission filter disposed between the first reaction chamber and theemission detecting sensor, wherein the filtering comprises allowinglight of one or more first wavelengths corresponding to the emittedfluorescent light, and filtering out light of one or more secondwavelengths, wherein the one or more first wavelengths are differentfrom the one or more second wavelengths.
 24. The method of claim 23,further comprising: filtering light from the excitation light sourcewith an excitation filter disposed between the first reaction chamberand the excitation light source, wherein the filtering comprisesallowing light of one or more third wavelengths configured to excite thefluorescent marker, and filtering out light of one or more fourthwavelengths, wherein the one or more third wavelengths are differentfrom the one or more fourth wavelengths.
 25. The method of claim 22,further comprising: detecting, by a temperature sensor, a temperatureassociated with the first reaction chamber; and logging, within amemory, a value indicating the detected temperature and a valuecorresponding to the emitted fluorescent light.